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The formation of amyloid fibril is associated with major human diseases, including Alzheimer's disease, prion diseases, and type 2 diabetes. Methods for efficient inhibition of amyloid fibril formation are therefore highly clinically important. A principal approach for the inhibition of amyloid formation is based on the use of modified molecular recognition elements. Here, we demonstrate efficient inhibition of amyloid formation of the type 2 diabetes-related human islet amyloid polypeptide (hIAPP) by a modified aromatic peptide fragment and a small aromatic polyphenol molecule. A molecular recognition assay using peptide array analysis suggested that molecular recognition between hIAPP and its core amyloidogenic module is mediated by aromatic rather than hydrophobic interactions. To study the possible effect of aromatic interactions on inhibition of hIAPP fibril formation, we have used peptide and small molecule inhibitors. The addition of a nonamyloidogenic peptide analogue of the core module NFGAILSS, in which phenylalanine was substituted with tyrosine (NYGAILSS), resulted in substantial inhibition of fibril formation by hIAPP. The inhibition was significantly stronger than the one achieved using a beta-sheet breaker-conjugated peptide NFGAILPP. On the basis of the molecular arrangement of the tyrosine-phenylalanine interaction, we suggest that the inhibition stems from the geometrical constrains of the heteroaromatic benzene-phenol interaction. In line with this notion, we demonstrate remarkable inhibition of hIAPP fibril formation and cytotoxicity toward pancreatic beta-cells by a small polyphenol molecule, the nontoxic phenol red compound. Taken together, our results provide further experimental support for the potential role of aromatic interactions in amyloid formation and establish a novel approach for its inhibition.

We present a combined experimental and theoretical investigation of the tendencies to form amyloid fibrils by a hexapeptide derivative of the human islet amyloid polypeptide, the NFGAIL (22-27) fragment and its mutants. We performed a complete alanine scan of this fragment and studied the capability of the wild-type and its mutant analogs to form ordered fibrils by ultrastructural and biophysical analyses. In parallel, we conducted a meticulous characterization of each sequence-complex at an atomistic level by performing nine independent molecular dynamics simulations for a total of 36 ns. These allowed us to rationalize the experimental observations and to establish the role of every residue in the fibrillogenesis. The main factor that determines the formation of regular fibrils is a coherent organization of the intersheet space. In particular, phenylalanine side chains cement the macromolecular assemblies due to their aromatic chemical character and restricted conformational flexibility when interacting with aliphatic residues.

The formation of amyloid fibrils by the human islet amyloid polypeptide is associated with type II diabetes. While it was previously suggested that the formed fibrils are toxic to pancreatic beta-cells due to membrane permeation activity, more recent studies suggested that protofibrillar assemblies have significantly higher potency in permeating lipid bilayers. Here, we specifically studied the membrane interaction activity of soluble and insoluble islet amyloid polypeptide assemblies at high temporal resolution. A colorimetric analysis using lipid/polydiacetylene (PDA) biomimetic vesicles clearly demonstrated the transient formation of soluble assemblies that strongly interact with the lipid vesicles. A peak in the level of membrane binding of the soluble fraction, as reflected by the colorimetric assay, was observed after incubation for approximately 1 h, followed by a decrease in the level of membrane interaction of the assemblies. The transient nature of the membrane-active assemblies was independently confirmed by a fluorescence quenching assay. Ultrastructural analysis using transmission electron microscopy provided morphological evidence of prefibrillar assemblies, supported the transient existence of membrane interacting soluble species, and facilitated observation of the non-membrane-active filaments in the solution. Taken together, our results provide experimental evidence for the formation of transient soluble prefibrillar assemblies which are highly membrane-active. The implications of these observations are discussed in light of designed fibrillization inhibitors.

The process of amyloid fibril formation by the human calcitonin hormone is associated with medullary thyroid carcinoma. Based on the effect of pH on the fibrillization of human calcitonin, the analysis of conformationally constrained analogues of the hormone, and our suggestion regarding the role of aromatic residues in the process of amyloid fibril formation, we studied the ability of a short aromatic charged peptide fragment of calcitonin (NH(2)-DFNKF-COOH) to form amyloid fibrils. Here, using structural and biophysical analysis, we clearly demonstrate the ability of this short peptide to form well ordered amyloid fibrils. A shorter truncated tetrapeptide, NH(2)-DFNK-COOH, also formed fibrils albeit less ordered than those formed by the pentapeptide. We could not detect amyloid fibril formation by the NH(2)-FNKF-COOH tetrapeptide, the NH(2)-DFN-COOH tripeptide, or the NH(2)-DANKA-COOH phenylalanine to the alanine analogue of the pentapeptide. The formation of amyloid fibrils by rather hydrophilic peptides is quite striking, because it was speculated that hydrophobic interactions might play a key role in amyloid formation. This is the first reported case of fibril formation by a peptide as short as a tetrapeptide and one of very few cases of amyloid formation by pentapeptides. Because the aromatic nature seems to be the only common property of the various very short amyloid-forming peptides, it further supports our hypothesis on the role of aromatic interactions in the process of amyloid fibril formation.